High oleic imidazolinone resistance sunflower

ABSTRACT

A sunflower seed having imidazolinone resistance and an oleic acid content of greater than 85 percent is provided. Sunflower cultivars designated E83329, OI1601A, OI2653R, and OI1601B and having high oleic acid and imidazolinone resistance, plants and seeds of the E83329, OI1601A, OI2653R, and OI1601B sunflower cultivars, methods for producing a sunflower plant produced by crossing the E83329, OI1601A, OI2653R, or OI1601B cultivar with itself or with another sunflower plant, and hybrid sunflower seeds and plants produced by crossing the E83329, OI1601A, OI2653R, or OI1601B cultivar with another sunflower line or plant are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/992,825, filed Mar. 28, 2008, pending, which application is anational entry of PCT/US2006/038034 filed Sep. 28, 2006 which claims thebenefit of U.S. Provisional Application No. 60/721,181 filed on Sep. 28,2005, the entire disclosure of each of which is hereby incorporatedherein by this reference.

TECHNICAL FIELD

This invention relates to a novel sunflower (Helianthus sp.) plant, toproducts obtained from the novel plant, and to methods of producing thesunflower products.

BACKGROUND

Sunflower is one of the few crop species that originated in NorthAmerica. It was probably domesticated by Native American tribes around1000 B.C. The first Europeans observed sunflower cultivated in manyplaces across North America, from southern Canada to Mexico. Sunflowerwas probably first introduced to Europe through Spain, eventuallyreaching Russia where it was extensively cultivated. Selection for highoil began in Russia in 1860 and resulted in oil content increasing from28 percent to 50 percent. These high-oil lines from Russia wereintroduced into the United States after World War II. The laterdiscovery of the male-sterile and restorer gene system made hybridsfeasible and increased commercial interest in the crop. Production ofsunflowers subsequently rose dramatically in the Great Plains states asmarketers found new niches for the seeds as an oil crop, a birdseedcrop, and as a human snack food.

The cultivated sunflower (Helianthus annuus L.) is a major worldwidesource of vegetable oil. In the United States, the major sunflowerproducing states are the Dakotas, Minnesota, Kansas, Colorado, Nebraska,Texas and California, although most states have some commercial acreage.Sunflower oil production in the United States was 2.26 million pounds in2003. Non-oil production was 406,000 pounds. Non-oil sunflowers averaged1,256 pounds per acre in 2003, while oil sunflowers had an average yieldof 1,206 pounds per acre in 2003.

Sunflowers are considered oilseeds, along with cottonseed, soybeans andcanola and the growth of sunflower as an oilseed crop has rivaled thatof soybean. The oil accounts for 80 percent of the value of thesunflower crop, as contrasted with soybean, which derives most of itsvalue from the meal. Sunflower oil is generally considered a premium oilbecause of its light color, high level of unsaturated fatty acids, lackof linolenic acid, bland flavor and high smoke points. The primary fattyacids in the oil are oleic and linoleic with the remainder consisting ofpalmitic and stearic saturated fatty acids.

Non-dehulled or partly dehulled sunflower meal has been substitutedsuccessfully for soybean meal in isonitrogenous (equal protein) dietsfor ruminant animals, as well as for swine and poultry feeding.Sunflower meal is higher in fiber, has a lower energy value and is lowerin lysine but higher in methionine than soybean meal. Protein percent ofsunflower meal ranges from 28 percent for non-dehulled seeds to 42percent for completely dehulled seeds.

In addition to its use in food and food products for humans and animals,sunflower oil also has industrial uses. It has been used in paints,varnishes and plastics because of good semidrying properties without thecolor modification associated with oils high in linolenic acid. It hasalso been used in the manufacture of soaps, detergents and cosmetics.The use of sunflower oil (and other vegetable oils) as a pesticidecarrier, and in the production agrichemicals, surfactants, adhesives,fabric softeners, lubricants and coatings has been explored.Considerable work has also been done to explore the potential ofsunflower as an alternate fuel source in diesel engines becausesunflower oil contains 93 percent of the energy of U.S. Number 2 dieselfuel (octane rating of 37). More recently, sunflower oil has beenproposed as a source of hydrogen for hydrogen fuel cells (BBC News, Aug.26, 2004).

Sunflower is an annual, erect, broadleaf plant with a strong taproot anda prolific lateral spread of surface roots. Stems are usually roundearly in the season, angular and woody later in the season, and normallyunbranched. The sunflower head is not a single flower (as the nameimplies) but is made up of 1,000 to 2,000 individual flowers joined at acommon receptacle. The flowers around the circumference are ligulate rayflowers without stamens or pistils; the remaining flowers are perfectflowers with stamens and pistils. Anthesis (pollen shedding) begins atthe periphery and proceeds to the center of the head. Since manysunflower varieties have a degree of self-incompatibility, pollenmovement between plants by insects is important, and bee colonies havegenerally increased yields.

The development of a cytoplasmic male-sterile and restorer system forsunflower has enabled seed companies to produce high-quality hybridseed. Most of these have higher yields than open-pollinated varietiesand are higher in percent oil. Performance of varieties tested overseveral environments is the best basis for selecting sunflower hybrids.The choice should consider yield, oil percent, maturity, seed size (fornon-oilseed markets), and lodging and disease resistance.

As a crop, sunflower yields are reduced, but rarely eliminated, byweeds, which compete with sunflower for moisture and nutrients andoccasionally for light. Sunflower is a strong competitor with weeds,especially for light, but does not cover the ground early enough toprevent weed establishment. Therefore, early season weed control isessential for good yields; successful weed control should include acombination of cultural and chemical methods. Almost all North Americansunflower plantings are cultivated and/or harrowed for weed control, andover ⅔ are treated with herbicides.

The imidazolinones are a class of herbicides that control a broadspectrum of weeds at low rates and are used throughout the world inlegumes, cereals, forests, and plantation crops. These herbicides arewidely used, not only because of their efficacy, but also because oftheir low mammalian toxicity and low environmental impact. Theavailability of imidazolinone-resistant crops offers many benefits andadvantages to the grower by allowing the development of a very flexibleweed management program. Because of the broad-spectrum activity andflexible application techniques of the imidazolinones, a weed managementprogram that utilizes a resistant crop can be based on the weeds thatneed to be controlled with less concern of the relative selectivity ofthe herbicide. Imidazolinone-resistant crops thus can be an effectiveweed management tool.

The features of commercially competitive plant varieties generallyinclude more than high yield with excellent standability. While yield isthe single most critical input that affects the crop producer's profit,producers expect consistency of yield from year to year, diseaseresistance, other value-added traits and, more recently, herbicideresistance. The addition of herbicide resistance has created bothopportunities as well as tremendous challenges in productionagriculture.

References: Putnam, et al. 1990, Sunflower in Alternative Field CropsManual, University of Wisconsin-Extension, Cooperative Extension;University of Minnesota: Center for Alternative Plant & Animal Products;Minnesota Extension Service; M. Boland and J. Stroade 2004, SunflowerIndustry Profile, Department of Agricultural Economics, Kansas StateUniversity; Agricultural Marketing Resource Center; Stephen Duke, Ed.,1996, Herbicide-Resistant Crops. Agricultural, Environmental, Economic,Regulatory, and Technical Aspects, CRC Press; U.S. Pat. No. 4,627,192;U.S. Pat. No. 5,276,264; and U.S. Pat. No. 6,388,113.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF SUMMARY

The following embodiments and aspects thereof are described andillustrated in conjunction with systems, tools and methods that aremeant to be exemplary and illustrative, not limiting in scope. Invarious embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

It is an aspect of the present invention to provide a hybrid sunflowerseed that has a total oleic acid content of at least 85.2 percent andhas resistance to imidazolinones.

It is another aspect of the present invention to provide new sunflowerplants that can be used efficiently to produce parent lines and hybridspossessing desirable agronomic traits in combination with high oleicacid content.

It is yet another aspect of the present invention to provide a methodfor producing a hybrid sunflower that is resistant to imidazolinones.

In accomplishing the foregoing aspects, there has been provided, inaccordance with the present invention, a sunflower seed having an oleicacid content of greater than 85.2 percent and tolerance toimidazolinone.

In accordance with yet another aspect of the present invention, therehas been provided a sunflower variety that has a total oleic acidcontent of at least 85.2 percent and has resistance to imidazolinones.

Other aspects, features, and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

In the description and examples that follow, a number of terms are usedherein. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

ALS inhibitor. As used herein, the “ALS inhibitor” means anyherbicidally effective form of sulfonylureas, triazolopyrimidinesulfonamides, imidazolinones or heteroaryl ethers including any saltthereof.

Allele. An “allele” is any of one or more alternative forms of a gene,all of which alleles relate to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. “Backcrossing” is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Commercially acceptable. The term “commercially acceptable” means asunflower variety or hybrid having a grain yield of greater than 2000pounds per acre over at least two years and ten environments.

FAME analysis. “Fatty Acid Methyl Ester (FAME) analysis” is a methodthat generates accurate quantification of the fatty acids that make upcomplex lipid classes.

Imidazolinone resistance (Imi). Resistance and/or tolerance is conferredby one or more genes that alter acetolactate synthase (ALS), also knownas acetohydroxy acid synthase (AHAS), allowing the enzyme to resist theaction of imidazolinone.

Oil content. Oil content is measured as percent of the whole dried seedand is characteristic of different varieties. It can be determined usingvarious analytical techniques such as NMR, NIR, and Soxhlet extraction.

Oleic acid content. Oleic acid is a monounsaturated fatty acid with thechemical formula C18H34O2. Its IUPAC name is cis-9-octadecenoic acid butit is commonly referred to as C18:1. Oleic acid content refers to thepercent of the total fatty acid fraction of sunflower oil that consistsof C18:1.

Percent oleic acid (OLE). Percent oil of the seed that is oleic acid.

Percent of total fatty acids. The percent of total fatty acids isdetermined by extracting a sample of oil from seed, producing the methylesters of fatty acids present in that oil sample and analyzing theproportions of the various fatty acids in the sample using gaschromatography. The fatty acid composition can also be a distinguishingcharacteristic of a variety.

Protein content. Protein content is measured as percent of whole driedseed and is characteristic of different varieties. This can bedetermined using various analytical techniques such as MR and Kjeldahl.

Resistance to lodging. Resistance to lodging measures the ability of avariety to stand up in the field under high yield conditions and severeenvironmental factors. A variety can have good (remains upright), fair,or poor (falls over) resistance to lodging. The degree of resistance tolodging is not expressed under all conditions but is most meaningfulwhen there is some degree of lodging in a field trial.

Single Gene Converted (Conversion). Single gene converted (conversion)plant refers to plants that are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique or via genetic engineering.

Total Saturated (TOTSAT). Total percent oil of the seed of the saturatedfats in the oil including C12:0, C14:0, C16:0, C18:0, C20:0, C22:0 andC24.0.

Mean Yield. Mean yield of all sunflower entries grown at a givenlocation.

Yield. Greater than 10 percent above the mean yield across ten or morelocations.

Check Average. The average for one or more check varieties or hybrids ina given location.

Prior to the instant invention, a sunflower variety has never beendeveloped having both high oleic acid oil and imidazolinone resistancecombined into one sunflower genotype. These traits have not previouslybeen combined in any commercial or wild-type sunflower. Having bothtraits in one sunflower variety substantially expands the utility of thecrop by providing the highly desirable high oleic acid oil and greaterflexibility in weed control.

All crop species are grown for the purpose of harvesting some product ofcommercial significance. Enhancement of productivity or yield of thatproduct is a major goal of most plant breeding programs. The highestpriority in most sunflower cultivar development programs is increasingseed yield. Seed yield is a quantitative character controlled by manygenes and strongly influenced by the environment. The heritability ofyield is the lowest and the most variable of the major agronomic traitsconsidered in cultivar development, with heritability estimates rangingfrom 3 to 58 percent. Yield is an example of a quantitative characterthat breeders attempt to improve beyond the level of that present incurrent cultivars. Disease resistance is required in most cases toprotect the yield potential of a cultivar.

It is a difficult challenge to incorporate an herbicide-resistant or-tolerant trait into high yielding cultivars. The difficulty isincreased by several orders of magnitude if a breeder attempts tocombine the herbicide resistance with high oleic acid oil into onecultivar. For a plant breeder to find a cultivar with sufficient merit(e.g., high yielding) to be increased and commercially distributed, itis necessary to make many crosses and grow thousands of experimentalgenotypes. The evaluation of so many genotypes is a huge task, andconsumes an enormous amount of the plant breeder's time and budget. Insome instances, it can take a decade or more from the time the originalcross is made to the time when a commercially viable genotype isidentified.

The effectiveness of selecting for genotypes with the traits of interest(e.g., high yield, herbicide resistance, high oleic acid oil) in abreeding program will depend upon: 1) the extent to which thevariability in the traits of interest of individual plants in apopulation is the result of genetic factors and is thus transmitted tothe progenies of the selected genotypes; and 2) how much the variabilityin the traits of interest (yield, herbicide resistance, high oleic acidoil) among the plants is due to the environment in which the differentgenotypes are growing. The inheritance of traits ranges from control byone major gene whose expression is not influence by the environment(i.e., qualitative characters) to control by many genes whose effectsare influenced by the environment (i.e., quantitative characters).Breeding for quantitative traits is further characterized by the factthat: 1) the differences resulting from the effect of each gene aresmall, making it difficult or impossible to identify them individually;2) the number of genes contributing to a character is large, so thatdistinct segregation ratios are seldom, if ever, obtained; and 3) theeffects of the genes may be expressed in different ways based onenvironmental variation. Therefore, the accurate identification oftransgressive segregants or superior genotypes with the traits ofinterest is extremely difficult and its success is dependent on theplant breeder's ability to minimize the environmental variationaffecting the expression of the quantitative character in thepopulation. The likelihood of identifying a transgressive segregant isgreatly reduced as the number of traits combined into one genotype isincreased. For example, if a cross is made between cultivars differingin three complex characters, such as yield, herbicide resistance andhigh oleic acid oil, it is extremely difficult to recover simultaneouslyby recombination the maximum number of favorable genes for each of thethree characters into one genotype. Consequently, all the breeder cangenerally hope for is to obtain a favorable assortment of genes for thefirst complex character combined with a favorable assortment of genesfor the second character into one genotype in addition to anherbicide-resistant gene.

The methods used in cultivar development programs and their probabilityof success are dependent on the number of characters to be improvedsimultaneously, such as, seed yield, disease resistance, andherbicide-resistant/tolerant traits. The proportion of desiredindividuals for multiple characters in a population is obtained bymultiplying together the proportion of desired individuals expected inthe population for each character to be improved. This assumes that thecharacters are inherited independently, i.e., are not geneticallylinked.

These principles can be applied not only to traditionally bred lines,but also to lines having one or more transgenes. Whether combiningdesirable traditional and transgenic traits via hybridization oftransgenic lines or cotransformation of multiple genes into one line,the combined effect on yield are likely to be multiplicative. Thelikelihood of identifying a line with a suitable combination of traitsis further reduced when considering the potential effects of a transgeneon the regulation of metabolism within a plant. For example, one canconsider the potential effect of genes conferring resistance toimidazolinones. The gene conferring this trait is a gene encoding amutant acetolactate synthase (ALS) enzyme. The ALS gene affects closelyrelated biochemical reactions in the synthesis of amino acids.

Acceptable lines have background genotypes that compensate for or aremainly unaffected by the perturbations caused by the introduced gene.When lines with acceptable herbicide resistance are combined by breedingwith lines with high oleic oil, the background genotypes that haveadjusted to the introduced or mutant genes are combined, and newgenotypes must be selected. The frequency of genotypes with suitableyield will be reduced accordingly. Therefore, it is an extremelydifficult hurdle to combine herbicide resistance with high yield and ahigh oleic acid content in a given sunflower variety or hybrid.Unexpectedly, the traits of imidazolinone resistance with high oleicacid content have been combined in a commercially acceptable cultivar inthe present invention. Once these traits have been combined in avariety, then the traits can be transferred to other geneticbackgrounds.

DETAILED DESCRIPTION OF THE INVENTION

Applicants have made a deposit of at least 2500 seeds of inbredsunflower plant OI1601A with the American Type Culture Collection(ATCC), Manassas, Va. 20110 USA under ATCC Accession No. PTA-9470.

Applicants have made a deposit of at least 2500 seeds of inbredsunflower plant OI1601B with the American Type Culture Collection(ATCC), Manassas, Va. 20110 USA under ATCC Accession No. PTA-9471.

Applicants have made a deposit of at least 2500 seeds of inbredsunflower plant E83329 with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA under ATCC Accession No. PTA-9473.

Applicants have made a deposit of at least 2500 seeds of inbredsunflower plant OI2653 with the American Type Culture Collection (ATCC),Manassas, Va. 20110 USA under ATCC Accession No. PTA-9472.

The seeds deposited with the ATCC on Sep. 8, 2008, were taken from adeposit maintained by Agrigenetics, Inc., d/b/a Mycogen Seeds, sinceprior to the filing date of this application. Access to this depositwill be available during the pendency of the application to theCommissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant(s) will maintain and make thisdeposit available to the public pursuant to the Budapest Treaty.

EXAMPLES

The following examples are provided to further illustrate the presentinvention and are not intended to limit the invention beyond thelimitations set forth in the appended claims.

Example 1: Sunflower Hybrid E83329 Having High Oleic Acid Content andImidazolinone Resistance

One example of imidazolinone resistance and high oleic acid content issunflower cultivar E83329. E83329 was developed through plant breedingand is stable and uniform. It is a high oleic sunflower that isresistant to imidazolinones. Some of the criteria used to select invarious generations include: seed yield, lodging resistance, emergence,disease tolerance and maturity. The hybrid has shown uniformity andstability, as described in the following variety descriptioninformation. The parent lines have been self-pollinated a sufficientnumber of generations with careful attention to uniformity of planttype. The hybrid has been increased with continued observation foruniformity. E83329 has the following morphologic and othercharacteristics.

TABLE 1 Plant: Height:   80 inches Number of leaves: 28 Leaf shape:Cordate Leaf length:   11 inches Leaf width: 10.7 inches Leaf marginindentation: Intermediate Leaf Attitude: Descending Days to Flower: 68Days to Maturity: 98 Ray flower color: Yellow Pappi: Green Headdiameter:   7 inches Head shape: Convex Head Attitude: Descending SeedNumber per head: 1675 Seed weight (g/200): 10 Harvest moisture(percent): 10.6 Yield (lbs/acre): 2910 Percent oil: 41 Oil Profile:Oleic: 86.9 percent C16:0: 4.05 percent C16:1: 0.18 percent C18:0: 2.93percent C18:2: 3.01 percent Saturated: 8.89 percent Imidazolinoneresistance: Excellent

In Table 2 that follows, select characteristics of E83329 are comparedwith commercial variety 7350.

TABLE 2 Trait E83329 7350 Days to Flower 68 64 Days to Maturity 98 96Height 80 inches 73 inches Number of Leaves 28 24 Head Diameter  7inches  8 inches Seed No. per Head 1675 1822 Seed Weight (g/200) 10 10Yield (lbs/acre) 2910 3034

In Table 3 that follows, the oil profiles of E83329 and commercialvariety 7350 are compared.

TABLE 3 Oil Trait E83329 7350 Total Percent Oil 41 percent 45.2 percentPercent Oleic 86.9 87.4 Percent C16:0 4.05 3.77 Percent C16:1 0.18 0.15Percent C18:0 2.93 3.11 Percent C18:2 3.01 2.93 Percent Saturated 8.898.62

In Table 4 that follows, the imidazolinone resistances of E83329 andcommercial variety 7350 are compared using a scale of 1 to 9 where 1 isexcellent resistance and 9 is poor resistance. Column 1 shows the dosageof herbicide applied and the time after application at which resistancewas measured. IMI is imidazolinone herbicide, 1×IMI is one times thestandard dosage of imidazolinone and so on.

TABLE 4 Imidazolinone Resistance E83329 7350 1 week after spray 1X IMI2.0 9.0 2X IMI 3.0 9.0 3X IMI 3.5 9.0 3 weeks after spray 1X IMI 1.0 9.02X IMI 1.0 9.0 3X IMI 1.0 9.0

In Table 5 that follows, the FAME analysis of E83329 is compared withthat of commercial variety 7350. Each figure is the percent of totalfatty acid oil.

TABLE 5 Oil Profile E83329 7350 C14:0 0.06 0.05 C16:0 4.05 3.77 C16:10.18 0.15 C18:0 2.93 3.11 C18:1 86.90 87.42 C18:2 3.01 2.93 C18:3 0.090.08 C20:0 0.35 0.34 C20:1 0.32 0.33 C20:2 0.01 0.01 C22:0 1.10 0.98C24:0 0.40 0.36 C24:1 0.01 0.01 TOTSAT 8.89 8.62

Example 2: Sunflower Cultivar OI1601A Having Imidazolinone Resistanceand High Oleic Acid Content

A second example of imidazolinone resistance and high oleic acid contentis sunflower cultivar OI1601A. OI1601A was developed through plantbreeding and is stable and uniform. It is a high oleic sunflower that isresistant to imidazolinones. Some of the criteria used to select invarious generations include: seed yield, lodging resistance, emergence,disease tolerance and maturity. The cultivar has shown uniformity andstability, as described in the following variety descriptioninformation. It has been self-pollinated a sufficient number ofgenerations with careful attention to uniformity of plant type. Thecultivar has been increased with continued observation for uniformity.OI1601A has the following morphologic and other characteristics.

TABLE 6 Plant: Height: 57 inches Number of leaves: 29   Leaf shape:Cordate Leaf length: 10.6 inches Leaf width: 10.2 inches Leaf marginindentation: Intermediate Leaf Attitude: Descending Days to Flower: 68  Days to Maturity: 95   Ray flower color: Yellow Pappi: Green Headdiameter: 7 inches Head shape: Convex Head Attitude: Descending Percentoil: 40.9 Oil Profile: Oleic: 88.95 percent C16:0:  3.53 percent C16:1: 0.13 percent C18:0:  2.49 percent C18:2:  2.98 percent Saturated:  7.55percent Imidazolinone resistance: Excellent

In Table 7 that follows, select characteristics of OI1601A are comparedwith commercial variety 7350.

TABLE 7 Trait OI1601A 7350 Days to Flower 68 64 Days to Maturity 95 96Height 57 inches 73 inches Number of Leaves 29 24 Head Diameter  7inches  8 inches Seed No. per Head 463 1822 Seed Weight (g/200) 12 10Yield (lbs/acre) 1,125 3034

In Table 8 that follows, the oil profiles of OI1601A and commercialvariety 7350 are compared.

TABLE 8 OI1 Trait OI1601A 7350 Total Percent Oil 40.9 45.2 Percent Oleic88.95 87.4 Percent C16:0 3.53 3.77 Percent C16:1 0.13 0.15 Percent C18:02.49 3.11 Percent C18:2 2.98 2.93 Percent Saturated 7.55 8.62

In Table 9 that follows, the imidazolinone resistances of OI1601A andcommercial variety 7350 are compared using a scale of 1 to 9 where 1 isexcellent resistance and 9 is poor resistance. Column 1 shows the dosageof herbicide applied and the time after application at which resistancewas measured. IMI is imidazolinone herbicide, 1×IMI is one times thestandard dosage of imidazolinone and so on.

TABLE 9 Imidazolinone Resistance OI1601A 7350 1 week after spray 1X IMI2.0 9.0 2X IMI 3.0 9.0 3X IMI 3.5 9.0 3 weeks after spray 1X IMI 1.0 9.02X IMI 1.0 9.0 3X IMI 1.0 9.0

Example 3: Sunflower Cultivar OI2653R Having Imidazolinone Resistanceand High Oleic Acid Content

A third example of imidazolinone resistance and high oleic acid contentis sunflower cultivar OI2653R. OI2653R was developed through plantbreeding and is stable and uniform. It is a high oleic sunflower that isresistant to imidazolinones. Some of the criteria used to select invarious generations include: seed yield, lodging resistance, emergence,disease tolerance and maturity. The cultivar has shown uniformity andstability, as described in the following variety descriptioninformation. It has been self-pollinated a sufficient number ofgenerations with careful attention to uniformity of plant type. Thecultivar has been increased with continued observation for uniformity.OI2653R has the following morphologic and other characteristics.

TABLE 10 Plant: Height: 62 inches Number of leaves: 24 Leaf shape:Cordate Leaf length: 11.7 inches Leaf width: 9.8 inches Leaf marginindentation: Intermediate Leaf Attitude: Descending Days to Flower: 74Days to Maturity: 102 Ray flower color: Yellow Pappi: Green Headdiameter: 4.5 inches Head shape: Flat Head Attitude: Descending Percentoil: 42.8 Oil Profile: Oleic: 89 percent C16:0: 3.4 percent C16:1: 0.11percent C18:0: 2.26 percent C18:2: 3.26 percent Saturated: 7.15 percentImidazolinone resistance: Excellent

In Table 11 that follows, select characteristics of OI2653R are comparedwith commercial variety 7350.

TABLE 11 Trait OI2653R 7350 Days to Flower 74 64 Days to Maturity 102 96Height 62 inches 73 inches Number of Leaves 24 24 Head Diameter 4.5inches  8 inches Seed No. per Head 510 1822 Seed Weight (g/200) 7 10Yield (lbs/acre) 400 3034

In Table 12 that follows, the oil profiles of OI2653R and commercialvariety 7350 are compared.

TABLE 12 Oil Trait OI2653R 7350 Total Percent Oil 42.8 percent 45.2percent Percent Oleic 89 87.4 Percent C16:0 3.4 3.77 Percent C16:1 0.110.15 Percent C18:0 2.26 3.11 Percent C18:2 3.26 2.93 Percent Saturated7.15 8.62

In Table 13 that follows, the imidazolinone resistances of OI2653R andcommercial variety 7350 are compared using a scale of 1 to 9 where 1 isexcellent resistance and 9 is poor resistance. Column 1 shows the dosageof herbicide applied and the time after application at which resistancewas measured. IMI is imidazolinone herbicide, 1×IMI is one times thestandard dosage of imidazolinone and so on.

TABLE 13 Imidazolinone Resistance O12653 R 7350 1 week after spray 1XIMI 2.0 9.0 2X IMI 3.5 9.0 3X IMI 3.5 9.0 3 weeks after spray 1X IMI 1.09.0 2X IMI 1.0 9.0 3X IMI 1.0 9.0

In Table 14 that follows, the FAME analysis of OI2653R is compared withthat of commercial variety 7350. Each figure is the percent of totalfatty acid oil.

TABLE 14 Oil Profile OI2653R 7350 C14:0 0.04 0.05 C16:0 3.40 3.77 C16:10.11 0.15 C18:0 2.26 3.11 C18:1 89 87.42 C18:2 3.26 2.93 C18:3 0.11 0.08C20:0 0.26 0.34 TOTSAT 7.15 8.62

Example 4: Sunflower Cultivar OI1601B Having Imidazolinone Resistanceand High Oleic Acid Content

A fourth example of imidazolinone resistance and high oleic content issunflower cultivar OI1601B. OI1601B was developed through plant breedingand is stable and uniform. It is a high oleic sunflower that isresistant to imidazolinones. Some of the criteria used to select invarious generations include: seed yield, lodging resistance, emergence,disease tolerance and maturity. The cultivar has shown uniformity andstability, as described in the following variety descriptioninformation. It has been self-pollinated a sufficient number ofgenerations with careful attention to uniformity of plant type. Thecultivar has been increased with continued observation for uniformity.OI1601B has the following morphologic and other characteristics.

TABLE 15 Plant: Height: 57 inches Number of leaves: 29 Leaf shape:Cordate Leaf length: 10.6 inches Leaf width: 0:2 inches Leaf marginindentation: Intermediate Leaf Attitude: Descending Days to Flower: 68Days to Maturity: 95 Ray flower color: Yellow Pappi: Green Headdiameter: 7 inches Head shape: Convex Head Attitude: Descending SeedNumber per head: 463 Seed weight (g/200): 12 Harvest moisture (percent):10 Yield (lbs/acre): 1,125 Percent oil: 40.9 Oil Profile: Oleic: 88.95percent C16:0: 3.53 percent C16:1: 0.13 percent C18:0: 2.49 percentC18:2: 2.98 percent Saturated: 7.55 percent Imidazolinone resistance:Excellent

In Table 16 that follows, the FAME analysis of OI1601B is compared withthat of commercial variety 7350. Each figure is the percent of totalfatty acid oil.

TABLE 16 Oil Profile OI1601B 7350 C14:0 0.04 0.05 C16:0 3.40 3.77 C16:10.11 0.15 C18:0 2.26 3.11 C18:1 89 87.42 C18:2 3.26 2.93 C18:3 0.11 0.08C20:0 0.26 0.34 TOTSAT 7.15 8.62

This invention is also directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant, wherein the first or second sunflower plant is thesunflower plant from the cultivar E833229, OI1601A, OI2653R, or OI1601B.Further, both first and second parent sunflower plants may be from thecultivar E833229, OI1601A, OI2653R, or OI1601B. Therefore, any methodsusing the cultivars E83329, OI1601A, OI2653R, or OI1601B are part ofthis invention: selfing, backcrosses, hybrid breeding, and crosses topopulations. Any plants produced using E83329, OI1601A, OI2653R, orOI1601B as a parent are within the scope of this invention.

Useful methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method suchas microprojectile-mediated delivery, DNA injection, electroporation andthe like. More preferably, expression vectors are introduced into planttissues using microprojectile delivery with a biolistic device orAgrobacterium-mediated transformation. Transformant plants obtained withthe protoplasm of the invention are intended to be within the scope ofthis invention.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes.” Over the last fifteen to twenty years, several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed variety or line.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid and can beused alone or in combination with other plasmids to provide transformedsunflower plants, using transformation methods as described below toincorporate transgenes into the genetic material of the sunflowerplant(s).

Expression Vectors for Sunflower Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent that maybe an antibiotic or an herbicide, or genes that encode an altered targetthat is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene under the control of plantregulatory signals, which confers resistance to kanamycin. Fraley etal., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983). Another commonlyused selectable marker gene is the hygromycin phosphotransferase gene,which confers resistance to the antibiotic hygromycin. Vanden Elzen etal., Plant Mol. Biol., 5:299 (1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant. Hayford et al., PlantPhysiol. 86:1216 (1988); Jones et al., Mol. Gen. Genet., 210:86 (1987);Svab et al., Plant Mol. Biol. 14:197 (1990); and Hille et al., PlantMol. Biol. 7:171 (1986). Other selectable marker genes confer resistanceto herbicides such as glyphosate, glufosinate or broxynil. Comai et al.,Nature 317:741-744 (1985); Gordon-Kamm et al., Plant Cell 2:603-618(1990); and Stalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987); Shah et al., Science 233:478 (1986); and Charest et al., PlantCell Rep. 8:643 (1990).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase and chloramphenicol acetyltransferase. R. A.Jefferson, Plant Mol. Biol. Rep. 5:387 (1987); Teeri et al., EMBO J.8:343 (1989); Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131 (1987);and DeBlock et al., EMBO J. 3:1681 (1984).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are available. Molecular Probes publication2908, Imagene Green™, p. 1-4 (1993), and Naleway et al., J. Cell Biol.115:151a (1991). However, these in vivo methods for visualizing GUSactivity have not proven useful for recovery of transformed cellsbecause of low sensitivity, high fluorescent backgrounds and limitationsassociated with the use of luciferase genes as selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells. Chalfieet al., Science 263:802 (1994). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Sunflower Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in certain tissues arereferred to as “tissue-specific.” A “cell type”-specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter that is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type-specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in sunflower. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequence thatis operably linked to a gene for expression in sunflower. With aninducible promoter, the rate of transcription increases in response toan inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system, whichresponds to copper (Mett et al., PNAS 90:4567-4571 (1993)), In2 genefrom maize, which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen. Genetics 227:229-237 (1991)), and Gatz etal., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10(Gatz et al., Mol. Gen. Genetics 227:229-237 (1991)). A particularlypreferred inducible promoter is a promoter that responds to an inducingagent to which plants do not normally respond. An exemplary induciblepromoter is the inducible promoter from a steroid hormone gene, thetranscriptional activity of which is induced by a glucocorticosteroidhormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in sunflower or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequence thatis operably linked to a gene for expression in sunflower.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985)) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)),ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989), andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)), pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)), MAS (Velten et al., EMBO J.3:2723-2730 (1984)), maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992), and Atanassova et al., Plant Journal 2(3):291-300 (1992)), Arabidopsis actin (Last and Gray, Plant Mol. Biol.12:655-666 (1989)), and pea plastocyanin promoter (McCabe et al., Theor.Appl. Genet. 99:587-592 (1999)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similar to the Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO 96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in sunflower.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence that is operably linkedto a gene for expression in sunflower. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983), and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)), a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729(1985), and Timko et al., Nature 318:579-582 (1985)), an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)), a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)), or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993)).

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample, Becker et al., Plant Mol. Biol. 20:49 (1992); P. S. Close,Master's Thesis, Iowa State University (1993); C. Knox et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley,” Plant Mol. Biol. 9:3-17 (1987); Lerner et al., Plant Physiol.91:124-129 (1989); Fontes et al., Plant Cell 3:483-496 (1991); Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991); Gould et al., J. Cell.Biol. 108:1657 (1989); Creissen et al., Plant J. 2:129 (1991); Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984); and Steifel, et al., Expression of a maize cellwall hydroxyproline-rich glycoprotein gene in early leaf and rootvascular differentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants that areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods,which are discussed, for example, by Heney and Orr, Anal. Biochem.114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is a sunflower plant. Inanother preferred embodiment, the biomass of interest is seed. For therelatively small number of transgenic plants that show higher levels ofexpression, a genetic map can be generated, primarily via conventionalRFLP, PCR and SSR analysis, which identifies the approximate chromosomallocation of the integrated DNA molecule. For exemplary methodologies inthis regard, see Glick and Thompson, Methods in Plant Molecular Biologyand Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance genes to engineer plants that are resistant to specificpathogen strains. See, for example, Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A gene conferring resistance to a pest, such as soybean cystnematode. See, e.g., PCT Application WO 96/30517 and PCT Application WO93/19181.

C. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

D. A lectin. See, for example, the disclosure by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

E. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

F. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor) and U.S. Pat. No.5,494,813 (Hepher and Atkinson, issued Feb. 27, 1996).

G. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

H. An insect-specific peptide or neuropeptide that, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

I. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

J. An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

K. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules that containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

L. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

M. A hydrophobic moment peptide. See PCT application WO 95/16776(disclosure of peptide derivatives of Tachyplesin, which inhibit fungalplant pathogens) and PCT application WO 95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

N. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci. 89:43 (1993),of heterologous expression of a cecropin-β, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

O. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

P. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

Q. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

R. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene that encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

S. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzymes as described, for example, by Lee etal., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase, bar, genes), andpyridinoxy or phenoxy proprionic acids and cyclohexones (ACCaseinhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 toShah, et al., which discloses the nucleotide sequence of a form of EPSPthat can confer glyphosate resistance. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession number 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061 to Comai. European patent application No. 0 333 033 to Kumadaet al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclosenucleotide sequences of glutamine synthetase genes that conferresistance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided inEuropean application No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy proprionic acids and cyclohexones, such as sethoxydim andhaloxyfop are the Accl-S1, Accl-S2 and Acct-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) or a benzonitrile (nitrilase gene). Przibila et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearoyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content—1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize for example, this could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele that is responsible for maize mutants characterized by lowlevels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Sunflower Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, B. R.Glick and J. E. Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,B. R. Glick and J. E. Thompson, Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation—One method for introducing anexpression vector into plants is based on the natural transformationsystem of Agrobacterium. See, for example, Horsch et al., Science227:1229 (1985). A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria that genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of the plant. See, for example,C. I. Kado, Crit. Rev. Plant Sci. 10:1 (1991). Descriptions ofAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provided by Gruber et al., supra, Miki et al., supra, andMoloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No.5,563,055 (Townsend and Thomas), issued Oct. 8, 1996.

B. Direct Gene Transfer—Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation. A generallyapplicable method of plant transformation is microprojectile-mediatedtransformation, wherein DNA is carried on the surface ofmicroprojectiles measuring 1 to 4 μm. The expression vector isintroduced into plant tissues with a biolistic device that acceleratesthe microprojectiles to speeds of 300 to 600 m/s, which is sufficient topenetrate plant cell walls and membranes. Sanford et al., Part. Sci.Technol. 5:27 (1987), J. C. Sanford, Trends Biotech. 6:299 (1988), Kleinet al., Bio/Technology 6:559-563 (1988), J. C. Sanford, Physiol. Plant7:206 (1990), Klein et al., Biotechnology 10:268 (1992). See also U.S.Pat. No. 5,015,580 (Christou, et al.), issued May 14, 1991; U.S. Pat.No. 5,322,783 (Tomes, et al.), issued Jun. 21, 1994.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985), and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of VIIth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell 4:1495-1505 (1992); and Spencer et al.,Plant Mol. Biol. 24:51-61 (1994).

Following transformation of sunflower target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic variety. The transgenic variety could then becrossed with another (non-transformed or transformed) variety in orderto produce a new transgenic variety. Alternatively, a genetic trait thathas been engineered into a particular sunflower line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple X by Y cross, or the process ofbackcrossing, depending on the context.

Tissue Culture of Sunflower

Further production of the OI1601A, OI2653R and OI1601B cultivars or theE83329 hybrid can occur by self-pollination or by tissue culture andregeneration. Tissue culture of various tissues of sunflower andregeneration of plants therefrom is known. For example, the propagationof a sunflower cultivar by tissue culture is described in, but notlimited to, any of the following: Shin et al., In Vitro Cellular andDevelopment Biology—Plant, 36:273-278 (2000); Hildebrandt and Riker,Amer. J. Bot., 34:421-427 (1947); Rogers et al., In Vitro, 6:463-7(1974); Fambrini et al., Ann. Bot., 92:145-152 (2003).

When the term “sunflower plant” is used in the context of the presentinvention, this also includes any single gene conversions of thatvariety. The term “single gene converted plant” as used herein refers tothose sunflower plants that are developed by a plant breeding techniquecalled backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single gene transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thevariety. The term “backcrossing” as used herein refers to the repeatedcrossing of a hybrid progeny back to the recurrent parent, i.e.,backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to the recurrentparent. The parental sunflower plant that contributes the gene for thedesired characteristic is termed the “nonrecurrent” or “donor parent.”This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and, therefore, does not recur. Theparental sunflower plant to which the gene or genes from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman &Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the originalvariety of interest (recurrent parent) is crossed to a second variety(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until asunflower plant is obtained, wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent, as determined at the 5percent significance level when grown in the same environmentalconditions.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic and, therefore, the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross. One ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance, itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. Single gene traits may or may notbe transgenic; examples of these traits include, but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Several of these single gene traits are described in U.S. Pat.Nos. 5,959,185, 5,973,234 and 5,977,445.

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of sunflowers andregeneration of plants therefrom is well known and widely published. Forexample, reference may be had to Mayor, et al., Plant Cell, Tissue andOrgan Culture, 72:99-103 (2003), and Baker et al., Plant Cell, Tissueand Organ Culture, 58:39-49 (1999). Thus, another aspect of thisinvention is to provide cells that, upon growth and differentiation,produce sunflower plants having the physiological and morphologicalcharacteristics of sunflower cultivars OI1601A, OI2653R, or OI1601B orthe sunflower hybrid E83329.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, pods, leaves,stems, roots, root tips, anthers, and the like. Means for preparing andmaintaining plant tissue culture are well known in the art. By way ofexample, a tissue culture comprising organs has been used to produceregenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445describe certain techniques, the disclosures of which are incorporatedherein by reference.

This invention is also directed to methods for producing a sunflowerplant by crossing a first parent sunflower plant with a second parentsunflower plant, wherein the first or second parent sunflower plant is asunflower plant of the variety OI1601A, OI2653R, or OI1601B or thehybrid E83329. Further, both first and second parent sunflower plantscan come from the sunflower variety OI1601A, OI2653R, or OI1601B or thehybrid E83329. Thus, any such methods using the sunflower varietyOI1601A, OI2653R, or OI1601B or the sunflower hybrid E83329 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using sunflower varietyOI1601A, OI2653R, or OI1601B or sunflower hybrid E83329, as a parent arewithin the scope of this invention, including those developed fromvarieties derived from sunflower variety OI1601A, OI2653R, or OI1601B orsunflower hybrid E83329. Advantageously, the sunflower variety may beused in crosses with other, different, sunflower plants to produce firstgeneration (F₁) sunflower hybrid seeds and plants with superiorcharacteristics. The variety of the invention may also be used fortransformation where exogenous genes are introduced and expressed by thevariety of the invention. Genetic variants created either throughtraditional breeding methods using variety OI1601A, OI2653R, or OI1601Bor hybrid E83329, or through transformation of variety OI1601A, OI2653R,or OI1601B or hybrid E83329 by any of a number of protocols known tothose of skill in the art are intended to be within the scope of thisinvention.

Deposits of the sunflower cultivars OI1601A, OI2653R, and OI1601B and ofsunflower hybrid E83329 are maintained by Dow Agrosciences atAgrigenetics, Inc. d/b/a Mycogen Seeds, Highway 75 North, Breckenridge,Minn. 56520. Access to these deposits will be available during thependency of this application to persons determined by the Commissionerof Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and35 USC 122. Upon allowance of any claims in this application, allrestrictions on the availability to the public of these varieties willbe irrevocably removed by affording access to deposits of at least 2,500seeds of each of the same varieties with the American Type CultureCollection, Manassas, Va.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is, therefore,intended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A sunflower plant, or parts thereof, having agene that confers tolerance to imidazolinone, wherein the sunflowerplant is selected from the sunflower cultivar consisting of OI1601A,OI1601B, OI2653R and E83329 that produces a sunflower seed having ATCCaccession No. PTA-9470, PTA-9471, PTA-9472 and PTA-9473, respectively,and wherein a seed oil of the sunflower seed comprises an oleic acidcontent of at least 85.2% based on total fatty acid content.
 2. Seed ofthe sunflower plant having a gene that confers tolerance toimidazolinone, wherein the seed is characterized by a seed oilcomprising an oleic acid content of at least 85.2% based on total fattyacid content, and wherein the seed is designated as E83329,representative samples having been deposited under ATCC accession No.PTA-9473.
 3. A tissue culture or a seed of the sunflower plant ofclaim
 1. 4. A method to produce a hybrid seed, wherein the methodcomprises crossing the plant of claim 1 with a second parent plant andharvesting the resultant hybrid seed.
 5. A hybrid plant produced bygrowing hybrid seed produced by the method according to claim 4, whereinsaid hybrid plant comprises said imidazolinone resistance gene.
 6. Aprocess for producing a sunflower oil, the process comprising:recovering sunflower oil from seed of the sunflower plant of claim
 1. 7.The sunflower plant or parts thereof of claim 1, wherein the sunflowerplant has a total saturated fatty acid content of less than 7.6 percentbased on total fatty acid content.